Luminol-enhanced Assay for Superoxide Anion (O2)

Stratagene introduces the LumiMax detection
assay, a simple and unique chemiluminescent-based method to qualitatively detect
the presence of superoxide anions in cell culture. In addition, the kit provides
a specific enhancement reagent to amplify chemiluminescent signal output that is
noncytotoxic and nondenaturing to live cells. Superoxide anion (O2), a potent oxidant, is a short-lived oxygen radical that is
released into the extracellular environment of stimulated leukocytes (monocytes,
macrophages, and polymorphonuclear leukocytes).1 Superoxide anion is
synthesized when NADPH-oxidase, a plasma membrane enzyme complex containing
cytochrome b 558, transfers an electron to molecular oxygen. Superoxide anions
are implicated in several key cellular processes, including oxidative stress
damage,2 tumor promotion,3 and, most recently, cell growth
and DNA synthesis via their involvement in the cell signaling pathway of
proto-oncogene ras.4

At present, superoxide may be detected by measuring the reduction of
exogenously supplied cytochrome c,5 the uptake of oxygen from
the medium (measured by the Clark electrode6), or luminol-mediated
chemiluminescence.7 The first method has limited sensitivity and is
complicated by the reoxidation of superoxide-reduced cytochrome c by
contaminants and cell lysis. The oxygen-uptake method is not only less sensitive
than cytochrome c reduction
but also requires a large number of cells (106)
per assay. The third method, luminol-mediated chemiluminescence, is the most
sensitive method of all; accurate readings can extend three orders of magnitude
over the signal range.

The Luminol Chemiluminescence Reaction

The chain of events for the luminol chemiluminescence (CL) assay is believed to involve cell surface receptors, such as those binding to complex immunoglobulin G (Fc receptors) or bacterial formylated peptides (f-met-leu-phe receptors), which are stimulated by their specific agonists. The activated receptor triggers the production or activation of NADPH oxidase, leading to the production of superoxide anion. Superoxide anion is subsequently released into the extracellular environment where it can oxidize luminol and available lipids and proteins. This oxidation results in chemiluminescence, a release of photons (light), which can be measured by a luminometer. Figure 1 illustrates Stratagenes enhanced chemiluminescent procedure for measuring superoxide anion.

When concentrations of superoxide anion are very low, assay sensitivity may
need to be enhanced. Some enhancement reagents, such as iodophenol, are
phenolic-based compounds. Unfortunately, such enhancers are toxic to live cells
and denaturing to some components of subcellular systems; hence, they cannot be
used for in situ assays. However, Stratagenes specific enhancement reagent
increases photon output but is noncytotoxic and does not denature cellular
components used in assaying live cell activity.

Assay for Superoxide Anions in U-937 cells

Cell cultures were provided with an analyte suspected of being able to
generate superoxide anion; luminol and enhancement reagents were then added.
Superoxide anion generated was measured as a result of luminol oxidation.

As a model, U-937 cells (monocytic-like, histocytic human lymphoma) were
cultured in RPMI supplemen
ted growth media with human gamma interferon (IFN-g)
to induce cell differentiation. U-937 cells were assayed for superoxide anions
after 2 to 5 days of differentiation with IFN-g.
After 2 days of differentiation, superoxide anion activity was minimal (data not
shown). However, after 3 days of differentiation, U-937 cells demonstrated
superoxide anion activity upon stimulation with 100 ng/ml phorbol 12-myristate
13-acetate (PMA), a protein kinase C activator and a potent stimulant of NADPH
oxidase. Reaction samples were aliquoted into polystyrene round-bottomed tubes
and placed into a luminometer to measure photon emission.

The luminometer was programmed to output raw data at 30-second counts.
Detection of superoxide anion with luminol, enhancer, or PMA independently
generated background numbers. Background values were determined against a
control flask containing cells without differentiation.

Figure 2 shows the relative light units (RLUs) recorded by the luminometer for samples incubated at varying time points after 3 days of differentiation and control samples containing induced cells with luminol plus PMA, induced cells with luminol plus enhancer, induced cells with PMA plus enhancer, and induced cells alone. The results indicated that luminol oxidation by superoxide anion is time dependent. Superoxide anion activity was also assayed after 4 and 5 days of differentiation with IFN-g. The resulting RLUs yielded lower total RLUs and required a longer incubation period before substantial RLU values were established. The decrease in photon output coupled with the longer incubation period may be attributed to the health of the cultured cells.

Superoxide Generating System: Xanthine-Xanthine Oxidase

To demonstrate that superoxide anions interact with and oxidize luminol, we assayed a known superoxide anion generating system compris
ed of xanthine oxidase and xanthine.8 In addition to assaying for superoxide anion activity, we suppressed its activity by introducing superoxide dismutase (SOD). Since SOD catalyzes the dismutation of O2 radicals into O2 and H2O2, the addition of SOD to xanthine-xanthine oxidase solution results in a decreased O2 concentration, which, in turn, leads to decreased oxidation of luminol or the chemiluminescent signal. Figure 3 shows the chemiluminescence assay of O2 generated by the control xanthine-xanthine oxidase reaction.

Conclusions

LumiMax is a simple, sensitive, and unique kit for researchers performing luminol-enhanced chemiluminescent assays. It has many applications including clinical assessment of leukocytes in patients who have altered oxidative means to control infections and biochemical research involving the analysis of leukocyte signal transduction pathwayscomponents comprising NADPH oxidase and requirements of the respiratory burst. In addition, the assay may be used to study the generation of oxygen radicals in AIDS patients9 and control their opportunistic infections; to detect, measure, or monitor cell aging or damage in selected agricultural crops;10 and to monitor cell damage by pesticides or environmental pollution.

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